Growth Response of Lactobacillus casei (ATCC 7469) to Riboflavin, FMN, and FAD.

Abstract

A method for determination of total tissue flavins (riboflavin, FMN, and FAD) involves extraction with 0.1 N HCI and microbiological assay of the extract using riboflavin as the standard ( 1 ) . The growth response of L a d obacillus casei (ATCC 7469) to these flavin compounds, based on acid production, has been noted to be identical(2).We present evidence here that shows that the growth response of this organism, measured turbidimetrically, differs significantly between the 3 flavin compounds, riboflavin, FMN, and FAD.Freshly prepared standard solutions of riboflavin, FMN, and FAD (Sigma. Chemical Co., St. Louis, Mo.) (100 ppmol/ml) were used for each assay. Ten separate assays were conducted. Six assays covered the range from 0 to 50 Fpmole per tube in 10 ppmole increments and 4 assays covered the range from 0 to 60 ppmole per tube in 20 ppmole increments.An aliquot of the standard solution was placed in an assay tube and sufficient water was added to give a volume of 1.5 ml. Doable strength assay media (Difco Laboratories, Detroit, Mi&) was then added to a final volume of 3 ml. All 3 compopunds were assayed concurrently in triplicate. The assay tubes were sterilized by autoclaving, cooled, and inoculated with a freshly prepared inoculum of L. casei (ATCC 7469) (3) a After incubation at 37°C for a period ranging from 21-22 hours, growth was measured turbidimetrically a t 650 qp. The results are expressed in terms of absmbancy units/ppmole flavin.Paper chromatographic analysis of the standard compounds was conducted in 3 of the experiments using the solvent systems described by Huennekens and Felton (4). Statistical analysis was by the "t" test as described by Snedecor ( 5). -* Abbreviations : FMN = flavin mononucleotide, FAD = flavin adenine dinuckotide. 0.2 '650MP 0. I 1 0 20 30 40 50 60 /L/LMOLES FLAVlN /ASSAY TUBE FIG. 1.The results (Fig. 1) show that there is a significant difference between riboflavin and FMN and a highly significant difference between ribo'flavin and FAD and between FMN and FAD with regard to the growth response of L. casei. A linear respnse of L. casei to all flavins tested was noted in all experiments.In the acid extraction of tissue Aavins there is a degradation of the FAD to FMN but little or nu breakdown of the FMN to riboflavin(6-8). Riboflavin per se accounts for a quantitatively insignificant amount of the total tissue flavin (8). The results presented here clearly show that the growth response to riboflavin and FMN is not the same when measured as described. The use, therefore, of riboflavin as a standard in the microbiological assay for tissue flavin extracted by acid would lead to quantitatively inaccurate results because very little of the extracted flavin is actually ribolflavin.

Riboflavin and other flavins (vitamin B2)

“…7469). Lactic acid bacteria cannot utilize FAD, and the growth response of L. casei, measured turbidimetrically, differs significantly between riboflavin and FMN (Langer and Charoensiri, 1966). As most of the vitamin B2 activity in foods is present in the form of FMN after acid extraction, it would be more accurate to use FMN as the standard in the microbiological assay instead of riboflavin.…”